Ⅰ、Antibody Fragments

Each complete immunoglobulin (IgG) molecule contains two heavy chains and two light chains connected by disulfide bonds. Antibody fragments (such as Fab, scFv and VHH) are small in size and have better tissue or tumor penetration than their full-length antibodies. Therefore, they have great prospects in immunotherapy, especially in solid tumors. In addition, they also have a short half-life and can be used as radioactive imaging agents. However, due to the lack of Fc region, they cannot cause Fc-mediated antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Ⅱ、Antibody Fab Fragment

2.1 Fab Fragment Structure

Fab fragment (Antigen-binding fragment), also known as antigen-binding fragment, is the region in the antibody structure that can bind to the antigen. It consists of a complete light chain (variable region and constant region) and a partial heavy chain structure (variable region and a constant region fragment). The light chain and the heavy chain are connected by a disulfide bond. It is small in size and has a molecular weight of 47-48 kDa.

2.2 Preparation of Fab Antibody

(1) Enzymatic hydrolysis: Fab can be obtained directly by enzymatic cleavage based on monoclonal antibody. Enzymatic hydrolysis usually uses enzymes such as papain or pepsin to degrade human immunoglobulin G to obtain products such as F(ab')2, Fab, and Fc fragments. The advantage of this method is that it is fast and simple, but the disadvantages are also obvious. First, it requires monoclonal antibody raw materials, which are generally limited in quantity and expensive. On the other hand, even after optimizing the enzymatic cleavage of the whole antibody, the obtained Fab often loses a certain degree of immunoreactivity.

(2) Preparation using expression system: The advantages of recombinant Fab are obvious. Since it does not have an Fc region, it does not require post-translational modification and glycosylation, and can be expressed in both prokaryotic and mammalian systems. The expression system is used to produce Fab fragments, usually using E. coli expression system and mammalian expression system. The E. coli expression system has the advantages of low production cost and fast production speed, but it is easy to form inclusion bodies and it is difficult to ensure activity after renaturation.

(3) Fab antibody library: Using phage display technology, first prepare a Fab fragment antibody library, and after several rounds of screening and enrichment, high-affinity Fab antibodies can be obtained. Theoretically, the mouse B cell antibody library is less than 10^8, and the human B cell antibody library is less than 10^12. The Fab combination antibody library can reach the level of 10^10-10^13, increasing the possibility of screening the ideal antibody.

III. Introduction to ScFv

ScFv antibodies contain variable heavy chain (VH) and variable light chain (VL). The two variable regions are connected by an artificially synthesized flexible peptide connector. This peptide connector is easily expressed in E.coil (E. coli), so we can use protein engineering to improve ScFv, such as increasing affinity or changing its specificity.

3.1 Phage Display Technology to Express ScFv

Total mRNA is extracted from immunized spleen B cells, and cDNA is obtained by reverse transcription. Then, the light chain variable region and heavy chain variable region DNA are amplified using cDNA as a template with specific primers. Then, the light chain variable region and heavy chain variable region are connected through Linker by overlapping PCR to obtain the total ScFv gene. The ScFv gene is connected to the pIT2 phage vector (containing resistance gene) and then transformed into E. coli (Ecoli TG1). In this way, a ScFv library is initially built. Ecoli TG1 containing the ScFv library is then co-cultured with KM13 helper phage to display ScFv on the phage surface, and the ScFv gene library is finally expanded by collecting phage particles. The product is obtained by enriching and screening the target ScFv phage particles and sent for sequencing.

3.2 Purification of Single-chain Antibodies

Single-chain antibodies lack the Fc fragment contained in the complete antibody, so the ordinary binding site (Protein G) cannot be used to purify the antibody. Usually, a HIS-tag is added to the C-terminus of the scFv fragment, and then the single-chain antibody can be purified using metal chelate affinity chromatography Ni-TED Purose 6 Fast Flow.

IV. Nanobodies (VHH)

Nanobodies (VHH) have the same structural domains as ordinary antibody VH, namely 4 conserved framework regions (FR1/2/3/4) and 3 complementary determining regions (CDR1/2/3). There are four highly conserved hydrophobic amino acid residues (V42, G49, L50 and W52) in FR2 of VH of ordinary antibodies, while in VHH antibodies, these four amino acids are replaced with hydrophilic amino acid residues (F42 or Y42, E49, R50 and G52), thus increasing the water solubility of nanobodies.

V. Advantages of Antibody Fragments

(1) Reduce nonspecific binding caused by Fc interactions (many cells have receptors that bind to the Fc region);

(2) Control Fc binding to protein A or protein G in immunoprecipitation and protein blotting experiments;

(3) More effective penetration of tissue sections, improving immunohistochemistry (IHC) staining;

(4) Reduce steric hindrance of large protein epitopes, and the sensitivity of antigen detection in solid-phase applications may be higher;

(5) Eliminate Fc-related effector functions (such as complement fixation) in antigen-antibody binding studies;

(6) Use X-ray crystallography or NMR to study immune recognition structural regions, which is easier to operate;

(7) The immunogenicity in endogenous experiments is lower than that of intact antibodies.

Tek Biotech has a complete recombinant antibody expression production platform and rich experience in recombinant protein and recombinant antibody expression. Based on phage display technology, we are able to provide customers with high-quality recombinant antibody preparation and screening services. At the same time, combined with large-scale recombinant protein fermentation platform, we are able to produce monoclonal antibodies including single-chain antibody (scFv), single-domain antibody, humanized antibody and Fc chimeric antibody for customers.